High-throughput screening assay for cholesterol inhibitors and inhibitors identified thereby

ABSTRACT

The present invention provides a high-throughput screening assay to identify test agents as cholesterol inhibitors via mutant NCP1 mammalian cells. Also provided are cholesterol inhibiting agents identified in accordance with this assay and methods for using such agents to inhibit cholesterol accumulation in cells.

INTRODUCTION

[0001] Work described herein was supported by funding from the NationalInstitutes of Health (Grant No. HL 36709) and the United StatesGovernment may have certain rights in this invention.

FIELD OF THE INVENTION

[0002] The present invention relates to a high-throughput screeningassay for identification of agents which inhibit or prevent theaccumulation of cholesterol in cells. More specifically, this cell-basedassay can be used to identify agents which block internalization ofplasma membrane cholesterol from entering the cell interior. Agentsidentified in accordance with this method are expected to be useful intreatment of cardiovascular as well as neurodegenerative diseasesassociated with cholesterol accumulation.

BACKGROUND OF THE INVENTION

[0003] Genetic disorders have provided important model systems toidentify factors and mechanisms involved in intracellular lipidmetabolism and trafficking. For example, human fibroblast (Hf) cellsfrom patients homozygous in familial hypercholesteremia have been usedto elucidate the low density lipoprotein receptor pathway involved inregulation of intracellular cholesterol metabolism (Brown, M. S. andGoldstein, J. L. Science 1986 232:34-47). Niemann-Pick type C diseasehas also provided important insights into cholesterol metabolism.Niemann-Pick type C disease is an autosomal recessive, neurovisceraldisorder that affects children who carry homozygous forms of the mutantNPC1 gene (Carstea et al. Science 1997 277:228-231) and causes deathbefore adulthood. Hf cells from patients with Niemann-Pick type Cdisease have been found to accumulate LDL-derived cholesterol asunesterified cholesterol in an intracellular compartment (Pentchev etal. Proc. Natl Acad. Sci. USA 1985 82:8247-8251; Pentchev et al. FASEBJ. 1987 1:40-45; and Liscum et al. J. Cell Biol. 1989 108:1625-1636).

[0004] The human NCP1 gene has been cloned, thus providing a betterunderstanding of Niemann-Pick type C disease at the molecular level(Cartsea et al. Science (1997) Proc. Natl Acad. Sci. USA 1997277:228-231). Final cloning work involved the identification of a 300 kbhuman genomic DNA containing the candidate NCP1 gene (Gu et al. Proc.Natl Acad. Sci. USA 1997 94:7378-7383). This unique DNA was identifiedby its ability to complement the defect of a previously isolated Chinesehamster ovary (CHO) cholesterol trafficking mutant, CT60 (Cadigan et al.J. Cell Biol. 110:295-308). The human NCP1 gene encodes an integralmembrane protein with 1278 amino acids and contains the “sterol-sendingdomains” (Watari et al. J. Biol. Chem. 1999 274:2111861-21866)identified in several other integral membrane proteins that respond toendoplasmic reticulum (ER) cholesterol.

[0005] In mammalian cells, low density lipoprotein (LDL) binds to itsreceptor and internalizes and enters the endosomes/lysosomes forhydrolysis of the lipid cargo cholesteryl esters (Brown, M. S. andGoldstein, J. L. Science 1986 232:34-47). Previously Niemann Pick type C(NPC) cells were believed to be defective in the movement of LDL-derivedcholesterol from the hydrolytic organelle to the plasma membrane,thereby leading to cholesterol accumulation in the lysosomes (Liscum etal. J. Cell Biol. 1989 108:1625-1636; Neufeld et al. 1996 J. Cell Biol.271:21604-21613). Evidence at the microscopic level, however,illustrated cholesterol to accumulate in the late endosomes of NPC cells(Neufeld et al. J. Biol. Chem. 1999 274:9627-9635; Kobayashi et al. Nat.Cell Biol. 1999 1:113-118) In additional studies, the movement ofLDL-derived cholesterol from the lysosomes to the plasma membrane inNPC-like cells was shown not to be defective (Lange et al. J. Biol.Chem. 1998 J. Biol. Chem 273:18915-18922). Using two independentlyisolated cholesterol-trafficking mutants defective in NPC1, namely CT60and CT43, a NPC1 stable transfectant and their parental cells, 25RA CHOcells, Cruz et al. recently disclosed evidence that NPCl is involved inpost-plasma membrane cholesterol trafficking (Cruz et al. J. Biol. Chem.2000 275(6):4013-4021). Specifically NPC1 was found to cycle cholesterolfrom an intracellular compartment to the plasma membrane or to theendoplasmic reticulum, but not prior to, newly hydrolyzed LDL-derivedcholesterol appears in the plasma membrane (Cruz et al. J. Biol. Chem.2000 275(6):4013-4021).

SUMMARY OF THE INVENTION

[0006] An object of the present invention is to provide ahigh-throughput screening assay to identify test agents as cholesterolinhibitors. In the assay of the present invention, mutant NCP1 mammaliancells, preferably CHO CT43 or CHO CT60 cells, are exposed to a testagent. The ability of the test agent to increase sterol efflux in themedia of the cells is evaluated, preferably via a pulse chase protocol.An increase in levels of sterol efflux in the media of the mutant NCP1cells exposed to the test agent as compared to mutant NCP1 cells notexposed to the test agent is indicative of the test agent being acholesterol inhibitor. Preferred cholesterol inhibitors of the presentinvention increase the sterol efflux level in the media of mutantmammalian NCP1 cells to the same level as observed in parenteral cells,preferably 25RA cells, not exposed to the test agent.

[0007] Another object of the present invention is to provide cholesterolinhibitors identified in accordance with this high-throughput screeningassay. Cholesterol inhibitors identified in accordance with the assay ofthe present invention are expected to be useful in preventing endogenouscholesterol accumulation observed in cardiovascular diseases as well asneurodegenerative disorders such as atherosclerosis. Such agents arealso useful in the treatment of Niemann Pick type C disease.

BRIEF DESCRIPTION OF THE FIGURES

[0008]FIG. 1 provides a working model of the intracellular traffickingof LDL-derived cholesterol in mammalian cells. In step 1, LDL crossesthe plasma membrane and enters the lysosomal/hydrolytic compartment.Initial movement of LDL cholesterol derived from the early hydrolyticdegradative organelles to the plasma membrane (step 2) does not requireNCP1. Upon reaching the plasma membrane, LDL-derived cholesterol isinternalized into an intracellular compartment, designated thecholesterol sorting compartment (step 3). NCP1 is involved in themovement of cholesterol from this intracellular compartment back to theplasma membrane (step 4 a) and to the endoreticulum for esterification(step 4 b).

DETAILED DESCRIPTION OF THE INVENTION

[0009] Lipoproteins are macromolecular complexes that carry hydrophobicplasma lipids, particularly cholesterol and triglyceride in the plasma.More than half of the coronary heart disease in the United States isattributable to abnormalities in the levels and metabolism of plasmalipids and lipoproteins. Premature coronary heart disease is sometimesrelated to mutations in the major genes involved in lipoproteinmetabolism. However, elevated lipoprotein levels in most patients withcoronary heart disease reflect the adverse impact of excess body weightand diets high in total and saturated fats. Elevated lipoprotein levelsin the brain have also been associated with neurodegenerative disorderssuch as Alzheimer's disease.

[0010] Treatment of elevated LDL cholesterol is typically either focusedat disease prevention or secondary treatment after complications haveoccurred. The rationale for primary prevention is based on a large bodyof evidence linking elevated levels of LDL cholesterol with an increasein coronary heart disease as well as clinical and experimental datademonstrating that reducing LDL cholesterol slows progression and mayactually induce regression of coronary heart disease.

[0011] Three classes of lipid-lowering agents are presently recommendedas first line therapy against hypercholesteremia. These include bileacid sequestrants or binding resins, niacin and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) inhibitors. Recent cloning of the cDNA forhuman-specific acyl coenzyme A:cholesterol acyltransferase (ACAT) hasalso enabled research efforts focused on development of ACAT inhibitorsfor the therapeutic prevention and treatment of human hypercholesteremiaand human atherosclerosis. However, there is a need for additionalcholesterol inhibiting agents as well as screening assays for theseagents.

[0012] The present invention provides a high-throughput screening assayfor use in evaluating and identifying test agents with the ability toinhibit internalization of LDL-derived cholesterol into an intracellularcompartment, designated the cholesterol sorting compartment (step 3 ofFIG. 1). Agents with this ability will block cholesterol accumulationwithin cells and will increase sterol efflux. Accordingly, such agentsare expected to be useful as cholesterol inhibitors in the treatment ofdiseases and disorders relating to over accumulation of cholesterol incells.

[0013] The assay of the present invention is a cell-based assay whichuses mammalian cells with a defective or mutant NCP1 gene, such as CHOCT43 or CT60 cells. CHO CT43 cells have been described and characterizedin detail in references by Cruz et al. (J. Biol. Chem. 2000275(6):4013-4021) and Cruz and Chang (J. Biol. Chem. 2000 275(52)41309-41316). CHO CT60 cells have been described by Cadigan et al. (J.Cell Biol. 1990 110:295-308) Culture conditions for growth of thesecells are set forth in Example 2. In the screening assay of the presentinvention, the mutant NCP1 cells are exposed to a test agent. Theability of the test agent to increase sterol efflux, preferably via apulse chase protocol, in the media of the cells is then evaluated. Anincrease in levels of sterol efflux in the media of mutant cells exposedto the test agent as compared to mutant cells not exposed to the testagent is indicative of the test agent being a cholesterol inhibitor. Ina preferred embodiment of the present invention, the mutant cellscomprise CHO CT43 or CT60 cells and levels of sterol efflux level in themedia of these cells when exposed to a test agent are compared to sterolefflux levels of parenteral 25RA cells not exposed to the test agent. Inthis embodiment, test agents which increase the level of sterol effluxin the media close to the level in 25RA cells are expected to be potentcholesterol inhibitors.

[0014] It is preferred that the screening assay of the present inventionbe performed in a microtiter well format so that multiple test agents atvarious concentrations can be evaluated simultaneously. In thisembodiment, mutant NPC1 cells are seeded into the wells of a microtiterplate. Sterol efflux in the media is preferably measured via a pulsechase protocol comprising detection of labeled cholesteryllinoleate-LDL. Examples of detectable labels include, but are notlimited to, radiolabels, fluorophores and enzymes. In addition to mutantNCP1 cells exposed to various test agents, it is preferred thatadditional wells containing only mutant cells and only parenteral cellsalso be included as negative and positive controls for the assay. Wellscontaining only mutant cells provide a negative control as sterol effluxlevels are expected to be low in these cells. These negative controlscan be used to determine increases in sterol efflux levels of the mutantcells upon exposure to the test agents. Increase in sterol efflux levelsupon exposure to the test agent as compared to the negative control isindicative of the test agent being a cholesterol inhibitors. Wellscontaining the parenteral cells provide a positive control of sterolefflux levels in normal cells. Test agents which increase sterol effluxlevels to levels of the positive control are expected to be veryeffective cholesterol inhibitors.

[0015] In a preferred embodiment, the mutant cells used in themicrotiter well format comprise CT43 cells or CT60 cells and are seededat approximately 3-4×10⁴ cells per well in medium A comprising Ham'sF-12, 10% FBS, and 10 μg/ml gentamycin). Control cells comprising theparenteral 25RA cells are seeded at approximately 1×10⁴ cells/well. Inthis embodiment, the medium is removed after one day, the cells arerinsed with phosphate buffered saline (PBS) and the medium is changed toMedium D comprising Ham's F12, 5% delipidated FBS, 10 mM Hepes, pH 7.4,35 μM oleic acid, and 10 μg/ml gentamycin. The CT43 or CT60 cells arethen incubated for an additional 36 hours. Prior to the pulse-chaseexperiment, cells are prechilled at 4° C. for 30 to 45 minutes. For thepulse, the cells are then incubated with [³H]cholesteryllinoleate-labeled LDL (Approximately 30 μg LDL/ml medium; specificactivity 30,000-50,000 cpm/μg protein, in 0.1 ml of medium D with sodiumcarbonate) at approximately 14° C. for about 4 hours. Cells are thenwashed and various test agents and/or various concentrations of a singletest agent are then added to the wells and the plates are incubated atapproximately 4° C. for about 1 hour. In a preferred embodiment the testagents are dissolved at high concentration in dimethyl sulfoxide (DMSO)so that the final concentration of DMSO in the assay is less than orequal to 1%. Typical concentrations of test agent examined range from 1to 100 μM. Following this incubation, the cells are chased with analiquot of medium D at 37° C. for various times ranging between 1 to 4hours. The cells are then subjected to 2% 2-hydroxypropylD-β-cyclodextrin (CD) in medium D at 37° C. for 30 minutes. TheCD-containing media and the cells can then be processed for radioactivecounting via direct counting as set forth in Example 3 or thin layerchromatography as set forth in Example 4. Using this microtiter wellformat of the assay of the present invention, CHO CT43 cells not exposedto any test agent were demonstrated to have significantly lower sterolefflux in their medium as compared to parenteral CHO 25RA cells.

[0016] Agents identified as cholesterol inhibitors in accordance withthe method of the present invention can block the internalization ofplasma membrane cholesterol from entering the cell interior therebycausing cholesterol to accumulate in the plasma membrane and promotingcholesterol efflux and stimulating reverse cholesterol transport invarious body cells. These agents are expected to slow the development ofatherosclerosis. Agents identified as inhibitors in accordance with themethod of the present invention can also block the internalization ofplasma membrane cholesterol in intestinal enterocytes, therebypreventing dietary cholesterol absorption. Such agents can also slowdown the accumulation of amyloid beta-peptides in the brain, therebyslowing down the symptoms of Alzheimer's disease. Accordingly, testagents identified as cholesterol inhibitors in accordance with the assayof the present invention are expected to be useful in preventing andtreating cardiovascular and neurodegenerative disease associated withover accumulation of cholesterol in cells. Such agents are also expectedto be useful in the treatment of Niemann Pick type C disease.

[0017] The following nonlimiting examples are provided to furtherillustrate the present invention.

EXAMPLES Example 1 Cell Lines

[0018] 25RA cells are a Chinese Hamster Ovary (CHO) cell line resistantto the cytotoxicity of 25-hydroxycholesterol containing a gain offunction mutation in the SREBP cleavage-activating protein (SCAP). CT43cells are derived from 25RA cells and are defective in NPC1.

Example 2 Cell Culture

[0019] CHO cells were seeded in medium A (Ham's F-12, 10% fetal bovineserum, and 10 μg/ml gentamicin) as monolayers at 37° C. with 5% CO₂ onday 1. On day 2, cells were incubated with medium D at 37° C. When usedat 37° C., medium D refers to Ham's F-12 with 5% delipidated fetalbovine serum, 35 μM oleic acid, 1.5 mM CaCl₂, and 10 μg/ml gentamicin;when used at 14° C., medium D refers to the same medium without sodiumbicarbonate and supplemented with 20 mM HEPES, pH 7. Al experiments wereconducted on day 4, when the cells were 80-90% confluent.

Example 3 Direct Counting

[0020] The cyclodextrin (CD)-containing media were transferred from thewells into scintillation vials and 3 ml of ECONOSCINT (NationalDiagnostics) was added to each vial and counted for radioactivity. Assoon as the CD-containing medium was removed, cells were washed with PBStwice, and 100 μl of 0.2 M of freshly prepared NaOH was added to eachwell to lyse the cells. After 30-45 minutes at room temperature, cellextracts were transferred into scintillation vials and 6.5 μl of 3M HCland 6.2 μl of 1 M KH₂PO₄, pH 7.0, were added to neutralize the cells.ECONOSCINT (3 ml) was then added to each vial, and processed forscintillation counting.

Example 4 Counting after Lipid Extraction and TLC Separation

[0021] The CD-containing medium was removed from the well and placedinto a 13×100 mm glass tube. After washing twice with PBS, cells werelysed with 100 μl of 0.2M NaOH, transferred into the glass tube, andneutralized with HCl/KH₂PO₄ by the procedure described in Example 3.Chloroform/methanol (2:1; 3 ml) was added to each sample and vortexedwell. H₂O (12 ml) was then added and the sample was vigorously vortexed.The samples were centrifuged at 1,000 g for 10 minutes, and the upperphase (aqueous phase) was removed. The remaining organic phase was driedunder N₂, and 100 μl of hexane (containing 1 mg/ml cold cholesterol) wasadded to each sample with vigorous vortex. The samples were spotted onSilica Gel TLC plate. The lipids were separated using the 90:10:1 ofpetroleum ether/ether/acetic acid solvent system. The plate wassubjected to I₂ staining to visualize the lipids, the band correspondingto cholesterol was scraped into the scintillation vial, solubilized with3 ml of BETAFLUOR (from National Diagnostics), and counted forradioactivity. % Efflux was calculated as the amount of [³H] cholesterolin medium divided by the sum of [³H] cholesterol in medium and in cellextract.

What is claimed is:
 1. A high-throughput. screening assay for identification of cholesterol inhibitors comprising exposing mutant NPC1 mammalian cells to a test agent, measuring a sterol efflux level in media of the mutant NPC1 mammalian cells exposed to the test agent, and comparing the measured level to a sterol efflux level in mutant NPC1 cells not exposed to the test agent, wherein an increase in the measured sterol efflux level in the mutant NPC1 cells exposed to the test agent as compared to the level in mutant NPC1 cells not exposed to the test agent is indicative of the test agent being a cholesterol inhibitor.
 2. The method of claim 1 wherein the mutant NPC1 cells comprise CHO CT43 or CT60 cells.
 3. The method of claim 1 wherein sterol efflux levels are measured via a pulse chase protocol.
 4. A high-throughput screening assay for identification of cholesterol inhibitors comprising exposing mutant NCP1 mammalian cells to a test agent, measuring a sterol efflux level in media of the mutant NCP1 mammalian cells exposed to the test agent, and comparing the measured level to a sterol efflux level in parenteral cells not exposed to the test agent, wherein a measured sterol efflux level in the mutant NCP1 cells exposed to the test agent equal to the sterol efflux level in parenteral cells test agent is indicative of the test agent being a cholesterol inhibitor.
 5. The method of claim 1 wherein the mutant NPC1 cells comprise CHO CT43 or CT60 cells and the parenteral cells comprise CHO 25RA cells.
 6. The method of claim 4 wherein sterol efflux levels are measured via a pulse chase protocol.
 7. A cholesterol inhibitor identified in accordance with the high-throughput screening assay of claim 1 or
 4. 8. A method of inhibiting over accumulation of cholesterol in cells comprising administering to the cells the cholesterol inhibitor of claim
 7. 9. A method of treating or preventing diseases or disorders relating to over accumulation of cholesterol in cells comprising administering to a patient the cholesterol inhibitor of claim
 7. 